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目的利用反向遗传技术分别对肠道病毒71型(EV71)强毒株SDLY107和弱毒株SDLY1的3C和3CD编码区进行置换,拯救重组病毒。方法使用PCR技术得到重组片段3C(1)-3D-3’UTR(107)和3CD(1)-3’UTR(107),并克隆入p M D19-T。利用双酶切、T4连接酶将两种重组3CD-3’UTR分别置换入本室构建并保存的EV71的SDLY107株的感染性c DNA克隆p M D19-T-107,得到重组p M D19-T-107后,将其线性化并转染入横纹肌肉瘤(RD)细胞,盲传得到重组病毒107(1-3C)和107(1-3CD)。对收获病毒进行鉴定。结果构建了重组EV71全长c DNA克隆;RNA转染并盲传至第3代,36 h后RD细胞出现细胞病变(CPE),48 h出现明显的CPE,成功拯救重组病毒SDLY107(1-3C)和SDLY107(1-3CD);重组病毒产生的CPE更接近SDLY107。结论成功构建了重组EV71反向遗传系统,拯救了重组病毒SDLY107(1-3C)和SDLY107(1-3CD),为进一步研究3C与3D蛋白在EV71致病机制中的作用提供基础。
Objective To reverse the 3C and 3CD coding regions of enterovirus 71 (EV71) virulent strain SDLY107 and virulent strain SDLY1 by reverse genetics to rescue the recombinant virus. Methods Recombinant fragments 3C (1) -3D-3’UTR (107) and 3CD (1) -3’UTR (107) were obtained by PCR and cloned into pM D19-T. The two recombinant 3CD-3’UTRs were respectively replaced by the T4 ligase into the infectious c DNA clone pM D19-T-107 of SDLY107 strain of EV71 that was constructed and stored in our laboratory. The recombinant pM D19- After T-107, it was linearized and transfected into rhabdomyosarcoma (RD) cells. The recombinant viruses 107 (1-3C) and 107 (1-3CD) were obtained by blind passage. Harvest the virus for identification. Results The full-length cDNA clone of recombinant EV71 was constructed. The RNA was transfected into the third passage blindly. The cytopathic effect (CPE) of RD cells was observed after 36 h. Significant CPE occurred at 48 h, and the recombinant virus SDLY107 (1-3C ) And SDLY107 (1-3CD); CPE produced by the recombinant virus is closer to SDLY107. Conclusion Recombinant EV71 reverse genetic system was successfully constructed and the recombinant viruses SDLY107 (1-3C) and SDLY107 (1-3CD) were rescued to provide a basis for further study on the role of 3C and 3D proteins in the pathogenesis of EV71.